AbstractIn this work, a self‐powered electrowetting valve (SPEV) driven by an energy‐harvesting triboelectric nanogenerator (TENG) is reported. The TENG (5 × 5 cm2) can produce an open‐circuit voltage of 380 V by applying a mechanical stimulus, which is much higher than the actuation voltage of the SPEV (130 V). Once actuated, the electrowetting valve can be instantly switched on at a response time of 0.18 s, allowing liquid reagent to flow through the valve. The SPEV can be used for simultaneous addition of multiple reagents in an enzyme‐linked immunosorbent assay on a paper‐based microfluidic analytical device (µPAD). This assay involves a chromogenic reaction that achieves effective detection of alpha‐fetoprotein, a critical tumor marker for early diagnosis of liver cancer. The SPEV reported in this work can be potentially used in other complex multiprocedure µPADs, which will potentially enable portable, accessible, and cost‐effective assays for early diagnosis, food safety, pollution detection, etc.